Cell-based high-throughput assays for fate decisions of human embryonic stem cell

描述(申请人提供)：在小鼠胚胎干细胞(MESC)被分离近20年后，1998年首次报道人类胚胎干细胞(HESCs)的来源，催生了hESC的研究领域。虽然这一领域还处于起步阶段，但人类胚胎干细胞已经被证明能够在培养中长期自我更新，并具有在体内发展为多种不同类型细胞的显著潜力(称为多能性)。因此，它们在理论上代表着取之不尽的前体细胞来源，用于治疗退行性、恶性或遗传性疾病，或因炎症、感染和创伤而造成的损伤。这种多能细胞被誉为治疗糖尿病、帕金森病、阿尔茨海默氏症、脊髓损伤、心力衰竭和骨髓衰竭的一种可能的方法。同时，人类胚胎干细胞是研究人类正常和异常发育的宝贵研究工具，可以作为开发和测试新药的平台。 我们的长期目标是定义管理人类胚胎干细胞命运决定的新条件和分子程序。如果我们最终要将这些细胞用于治疗，这些知识是必不可少的。目前对hESC的长期自我更新或谱系特异性分化的理解非常初级。为了解决这些问题，我们筛选了一小部分药物抑制剂，并确定了一种Ser-Thr蛋白激酶，作为控制hESCs未分化生长的关键调控分子。这一初步研究为大规模文库筛选在人类胚胎干细胞研究中的应用提供了概念验证。因此，我们将开发基于细胞的高通量分析方法，通过建立含有增强型绿色荧光蛋白(EGFP)或荧光素酶报告的hESCs来实现多能性或定向分化。这项研究的结果将为大规模的文库筛选工作奠定基础，以寻找影响hESCs命运决定的分子。因此，我们预计这些检测将为科学界提供新的工具来研究决定hESC命运的分子机制，并可能有助于有效的组织修复和再生策略。人类胚胎干细胞(HESCs)在了解人类早期发育以及寻找和测试治疗心血管疾病、神经退行性疾病和糖尿病等多种疾病的新药方面具有很大的前景。为了实现hESCs的治疗潜力，我们提出了建立高通量分析的实验，以在分子水平上确定这些细胞如何在培养中自我更新并分化为体内特定类型的细胞。这些检测也可能有助于产生足够的分化细胞，使我们能够评估hESCs在疾病的临床前模型中的治疗潜力，并为药物开发提供平台。

DESCRIPTION (provided by applicant): Nearly 20 years after murine embryonic stem cells (mESC) were isolated, the first report of the derivation of human embryonic stem cells (hESCs) in 1998 spawned the field of hESC research. Although this field is only in its infancy, hESCs have already been shown to be capable of long-term self-renewal in culture and have remarkable potential to develop into many different cell types in the body (known as pluripotency). They therefore represent a theoretically inexhaustible source of precursor cells to treat degenerative, malignant, or genetic diseases, or injury due to inflammation, infection, and trauma. This pluripotent cell has been hailed as a possible means for treating diabetes, Parkinson's disease, Alzheimer's, spinal cord injury, heart failure, and bone marrow failure. Meanwhile, hESCs are an invaluable research tool to study human development, both normal and abnormal, and can serve as a platform to develop and test new drugs. Our long-term goal is to define new conditions and molecular programs that govern fate decisions of hESCs. The knowledge is essential if we are ultimately to use these cells for therapy. The current understanding of hESC long-term self-renewal or lineage-specific differentiation is extremely rudimentary. As an attempt to address these questions, we screened a small collection of pharmacological inhibitors and identified a protein Ser-Thr kinase, as a key regulatory molecule that controls the undifferentiated growth of hESCs. This pilot study provided proof-of-concept for applying large-scale library screening to the study of hESCs. Accordingly, we will develop cell-based high-throughput assays by establishing hESCs containing enhanced green fluorescence protein (EGFP) or luciferase reporters for pluripotentcy or for directed differentiation. The results from the study will set the stage for large-scale library-screening efforts for searching molecules that influence the fate decisions of hESCs. Therefore, we expect that these assays will provide new tools for the scientific community to study the molecular mechanisms underlying hESC fate determination and may contribute to effective strategies for tissue repair and regeneration. Human embryonic stem cells (hESCs) hold considerable promise for understanding early human development and for finding and testing new drugs for a vast number of conditions, including cardiovascular diseases, neurodegenerative processes and diabetes. To realize the therapeutic potential of hESCs, we present experiments to establish high throughput assays for determining how, at the molecular level, these cells self-renew in culture and differentiate into a specific type of cells in the body. These assays may also facilitate production of sufficient differentiated cells to allow us to assess the therapeutic potential of hESCs in preclinical models of diseases, and to offer platforms for drug development.